Continuing studies based on dihydroquinoline glucocorticoid receptor agonists lead to the discovery of a series of C4-oxime analogs. Representative compounds exhibited potent transrepression activity with minimal transactivation of phosphoenolpyruvate caboxykinase (PEPCK), a key protein in the gluconeogenesis pathway. These compounds represent promising leads in identifying GR agonists with high anti-inflammatory activity and attenuated potential for glucose elevation.

Continuing studies on tetrahydroquinoline glucocorticoid receptor anti-inflammatory agents lead to the identification of several tetrahydroquinolin-3-yl carbamates that exhibited steroid-like activity in in vitro transrepression assays with reduced transactivation of phosphoenol pyruvate carboxykinase (PEPCK), a key enzyme in the gluconeogenesis pathway.

Abstract 3391 Granulocyte colony stimulating factor (GCSF) is the essential cytokine for the regulation of neutrophilic granulocytes. Binding of GCSF to its receptor (GCSFR) triggers receptor dimerization, leading to activation of JAK1 and JAK2, phosphorylation of GCSFR, STAT3, STAT5, and Ras/mitogen-activated protein kinase (MAPK), and results in proliferation and differentiation of granulocytic cells. Recombinant human GCSF (rhGCSF) is used successfully to alleviate chemotherapy-induced neutropenia, neutropenia associated with hematopoietic stem cell transplantation, and severe chronic neutropenia. A small molecule oral GCSFR agonist may offer a safer and more convenient alternative to the current injectable rhGCSF therapy. Whereas a previous effort to identify small-molecule mimetics of GCSF found SB-247464 that selectively activated the murine GCSFR, no small-molecule human GCSF mimetics have been developed. Recently, we have discovered a series of novel non-peptidyl small molecules that selectively activate human GCSFR (hGCSFR) function, and may provide a significant innovation in the treatment of neutropenia. In cells transiently transfected with an hGCSFR expression vector and a STAT3-responsive luciferase reporter, a lead compound, LG7455, activates luciferase expression with an efficacy of 50% relative to rhGCSF, and potency (EC50) of 100 nM. LG7455 also activates luciferase expression in cells transfected with hGCSFR and a STAT5-responsive luciferase reporter (65%, 40 nM EC50). The activity of LG7455 is dependent on the expression of hGCSFR, and LG7455 is not active in luciferase assays when human thrombopoietin receptor (hTPOR) or erythropoietin receptor (hEPOR) is expressed. In UT-7 cells made responsive to GCSF by stable transfection of hGCSFR (UTP-hGCSFR), LG7455 stimulated cell growth and increased the phosphorylation of STAT3 and STAT5. LG7455 did not increase growth of TPO- or EPO-responsive UT-7 cells. In CD34 positive human bone marrow hematopoietic cells (BM-HCs), LG7455, increased the percentage of cells positive for the granulocyte-specific marker CD15 (FUT4). The effect of LG7455 in BM-HCs was additive to the effect of rhGCSF. LG7455 is active in luciferase assays with expressed cynomolgus monkey GCSFR, but not mouse, guinea pig or rabbit GCSFR. Similar to what has been demonstrated for small-molecule human TPOR agonists such as eltrombopag, the activity of LG7455 is dependent on a specific residue in the hGCSFR transmembrane domain. When histidine 627 (His-627) in hGCSFR is changed to asparagine present at a similar location in the mouse GCSFR (Asp-602), unlike rhGCSF, LG7455 is no longer active. LG7455 is active, however, on mouse GCSFR with Asp-602 replaced by His. In radioligand-binding experiments using UTP-hGCSFR cells, LG7455 did not displace [125I]rhGCSF, however binding of [125I]rhGCSF was augmented in a concentration dependent manner consistent with allosteric receptor modulation. These data demonstrate that LG7455 is a novel small-molecule selective hGCSFR agonist that activates the receptor in a manner distinct from GCSF and similar to the mechanism of small-molecule hTPOR agonists. Further optimization of the LG7455 chemical series should provide orally-available molecules to treat neutropenia with improved safety and convenience compared to current injectable rhGCSF.Disclosures: Marschke: Ligand Pharmaceuticals: Employment. Rungta: Ligand Pharmaceuticals: Employment. Slavin: Ligand Pharmaceuticals: Employment. Sanders: Ligand Pharmaceuticals: Employment. Roach: Ligand Pharmaceuticals: Employment. Pickens: Ligand Pharmaceuticals: Employment. Shen: Ligand Pharmaceuticals: Employment. van Oeveren: Ligand Pharmaceuticals: Employment. Hong: Ligand Pharmaceuticals: Employment. Sun: Ligand Pharmaceuticals: Employment. Bissonnette: Ligand Pharmaceuticals: Employment. Syka: Ligand Pharmaceuticals: Employment. Zhi: Ligand Pharmaceuticals: Employment.

Abstract Abstract 1565 Erythropoietin (EPO) acts on the homodimeric EPO receptor (EPOR) to stimulate proliferation of erythroid progenitor cells and induce their survival and differentiation into red blood cells. Various recombinant human EPO derivatives, also known as erythropoiesis-stimulating agents (ESAs), are marketed or in clinical development for the treatment of anemia due to renal failure or cancer chemotherapy. However, ESA treatment is associated with an increased risk of adverse cardiovascular complications in patients with kidney disease, and may be related to an increase in mortality in cancer patients, when it is used to increase hemoglobin levels above 13.0 g/dl. We have identified a series of novel non-peptidyl small molecules that selectively activate EPOR function, which may provide a unique therapeutic opportunity in the treatment of anemia. In CD34 positive human bone marrow hematopoietic cells (BM-HCs), a representative analog, LG5640, potently (2 nM EC50) increased the percentage of cells positive for the erythrocyte-specific marker CD235a (glycophorin A) with an efficacy partial (42%) to the maximal effect of EPO (3 U/ml), but greater than the efficacy of the normal serum EPO concentration (∼0.01 U/ml). The erythropoietic effect of LG5640 in BM-HCs was additive to the effect of EPO. LG5640 stimulated the expression of several EPO responsive genes in CD34 positive BM-HCs, including hemoglobin α, EPOR and the anti-apoptotic protein BCL2L1. In addition, LG5640 stimulated the formation of BFUe colonies with partial efficacy (30%) when incubated with BM-HCs for 14 days. The effect of LG5640 on BM-HCs was specific for the erythroid lineage. LG5640 did not increase the percentage of BM-HCs positive for the megakaryocyte marker CD41 or the granulocyte marker CD15. Using human cell lines, we have determined that the action of LG5640 is dependent on EPOR and involves the selective activation of the PI3K/AKT-GATA1 signaling pathway. In the human EPO-dependent cell line UT7EPO, LG5640 blocked apoptosis induced by EPO withdrawal (10 nM EC50), and stimulated the expression of BCL2L1 with an efficacy comparable to EPO. LG5640 stimulated the phosphorylation of EPOR, PI3K, and GATA1, and induced the binding of GATA1 to DNA. Incubation of UT7EPO cells with the PI3K inhibitor LY294002 blocked the effect of LG5640 on cell survival. However, LG5640 did not stimulate phosphorylation of STAT5 or ERK/MAPK, or induce STAT5 DNA binding. LG5640 did not block apoptosis or stimulate BCL2L1 expression in the GM-CSF- and TPO-responsive human leukemia Mo7e cells that lack EPOR. Furthermore, transfection of UT7EPO cells with EPOR- and GATA1-specific siRNAs blocked the activity of LG5640. These data demonstrate that LG5640 is a novel small molecule selective EPOR agonist that unlike other ESAs selectively activates the EPOR/PI3K/GATA1 signal transduction pathway resulting in survival and differentiation of BM-HCs into erythrocytes, possibly through uniquely altering the conformation of the homodimeric EPOR. The selective agonists display an efficacy partial to the maximal effect induced by EPO, and lack excessive erythropoietic stimulation that may possibly contribute to the adverse effects of ESAs. Based on the novel profile of the series, several lead compounds that increase the percentage of CD235a positive BM-HCs with nanomolar potency, and display oral bioavailability in the rat and monkey, have been identified as potential preclinical development candidates. Disclosures: Bissonnette: Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Rungta:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hudson:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Roach:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hong:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Sun:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Hu:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Ward:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Luo:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Sanders:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Syka:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Slavin:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Zhi:Ligand Pharmaceuticals Inc: Employment, Equity Ownership. Marschke:Ligand Pharmaceuticals Inc: Employment, Equity Ownership.

Treatment of inflammation is often accomplished through the use of glucocorticoids. However, their use is limited by side effects. We have examined the activity of a novel glucocorticoid receptor ligand that binds the receptor efficiently and strongly represses inflammatory gene expression. This compound has potent antiinflammatory activity in vivo and represses the transcription of the inflammatory cytokine monocyte chemoattractant protein-1 and induces the antiinflammatory cytokine IL-10. The compound demonstrates differential gene regulation, compared with commonly prescribed glucocorticoids, effectively inducing some genes and repressing others in a manner different from the glucocorticoid prednisolone. The separation between the antiinflammatory effects of LGD-5552 and the side effects commonly associated with glucocorticoid treatment suggest that this molecule differs significantly from prednisolone and other steroids and may provide a safer therapeutic window for inflammatory conditions now commonly treated with steroidal glucocorticoids.

Structure-activity relationship studies centered around 3'-substituted (Z)-5-(2'-(thienylmethylidene))1,2-dihydro-9-hydroxy-10-methoxy-2,2,4-trimethyl-5H-chromeno[3,4-f]quinolines are described. A series of highly potent and efficacious selective glucocorticoid receptor modulators were identified with in vitro activity comparable to dexamethasone. In vivo evaluation of these compounds utilizing a 28 day mouse tumor xenograft model demonstrated efficacy equal to dexamethasone in the reduction of tumor volume.

Specific retinoid X receptor (RXR) agonists, such as LG100268 (LG268), and the thiazolidinedione (TZD) PPARgamma agonists, such as rosiglitazone, produce insulin sensitization in rodent models of insulin resistance and type 2 diabetes. In sharp contrast to the TZDs that produce significant increases in body weight gain, RXR agonists reduce body weight gain and food consumption. Unfortunately, RXR agonists also suppress the thyroid hormone axis and generally produce hypertriglyceridemia. Heterodimer-selective RXR modulators have been identified that, in rodents, retain the metabolic benefits of RXR agonists with reduced side effects. These modulators bind specifically to RXR with high affinity and are RXR homodimer partial agonists. Although RXR agonists activate many heterodimer partners, these modulators selectively activate RXR:PPARalpha and RXR:PPARgamma, but not RXR:RARalpha, RXR:LXRalpha, RXR:LXRbeta, or RXR:FXRalpha. We report the in vivo characterization of one RXR modulator, LG101506 (LG1506). In Zucker fatty (fa/fa) rats, LG1506 is a potent insulin sensitizer that also enhances the insulin-sensitizing activities of rosiglitazone. Administration of LG1506 reduces both body weight gain and food consumption and blocks the TZD-induced weight gain when coadministered with rosiglitazone. LG1506 does not significantly suppress the thyroid hormone axis in rats, nor does it elevate triglycerides in Sprague Dawley rats. However, LG1506 produces a unique pattern of triglycerides elevation in Zucker rats. LG1506 elevates high-density lipoprotein cholesterol in humanized apolipoprotein A-1-transgenic mice. Therefore, selective RXR modulators are a promising approach for developing improved therapies for type 2 diabetes, although additional studies are needed to understand the strain-specific effects on triglycerides.

LSN862 is a novel peroxisome proliferator-activated receptor (PPAR)alpha/gamma dual agonist with a unique in vitro profile that shows improvements on glucose and lipid levels in rodent models of type 2 diabetes and dyslipidemia. Data from in vitro binding, cotransfection, and cofactor recruitment assays characterize LSN862 as a high-affinity PPAR gamma partial agonist with relatively less but significant PPAR alpha agonist activity. Using these same assays, rosiglitazone was characterized as a high-affinity PPAR gamma full agonist with no PPAR alpha activity. When administered to Zucker diabetic fatty rats, LSN862 displayed significant glucose and triglyceride lowering and a significantly greater increase in adiponectin levels compared with rosiglitazone. Expression of genes involved in metabolic pathways in the liver and in two fat depots from compound-treated Zucker diabetic fatty rats was evaluated. Only LSN862 significantly elevated mRNA levels of pyruvate dehydrogenase kinase isozyme 4 and bifunctional enzyme in the liver and lipoprotein lipase in both fat depots. In contrast, both LSN862 and rosiglitazone decreased phosphoenol pyruvate carboxykinase in the liver and increased malic enzyme mRNA levels in the fat. In addition, LSN862 was examined in a second rodent model of type 2 diabetes, db/db mice. In this study, LSN862 demonstrated statistically better antidiabetic efficacy compared with rosiglitazone with an equivalent side effect profile. LSN862, rosiglitazone, and fenofibrate were each evaluated in the humanized apoA1 transgenic mouse. At the highest dose administered, LSN862 and fenofibrate reduced very low-density lipoprotein cholesterol, whereas, rosiglitazone increased very low-density lipoprotein cholesterol. LSN862, fenofibrate, and rosiglitazone produced maximal increases in high-density lipoprotein cholesterol of 65, 54, and 30%, respectively. These findings show that PPAR gamma full agonist activity is not necessary to achieve potent and efficacious insulin-sensitizing benefits and demonstrate the therapeutic advantages of a PPAR alpha/gamma dual agonist.

The synthesis and in vitro characterization of novel RXR-selective ligands possessing various substituted 1-benzofuran or 1-benzothiophene moieties are described.

To understand the species selectivity in a series of alpha-methyl-alpha-phenoxy carboxylic acid PPARalpha/gamma dual agonists (1-11), structure-based molecular modeling was carried out in the ligand binding pockets of both human and mouse PPARalpha. This study suggested that interaction of both 4-phenoxy and phenyloxazole substituents of these ligands with F272 and M279 in mouse PPARalpha leads to the species-specific divergence in ligand binding. Insights obtained in the molecular modeling studies of these key interactions resulted in the ability to convert a human-selective PPARalpha agonist to a human and mouse dual agonist within the same platform.

The design and synthesis of the dual peroxisome proliferator activated receptor (PPAR) alpha/gamma agonist (S)-2-methyl-3-[4-[2-(5-methyl-2-thiophen-2-yl-oxazol-4-yl)ethoxy]phenyl]-2-phenoxypropionic acid (2) for the treatment of type 2 diabetes and associated dyslipidemia are described. 2 possesses a potent dual hPPAR alpha/gamma agonist profile (IC(50) = 28 and 10 nM; EC(50) = 9 and 4 nM, respectively, for hPPARalpha and hPPARgamma). In preclinical models, 2 substantially improves insulin sensitivity and potently reverses diabetic hyperglycemia while significantly improving overall lipid homeostasis.

Benzofused heterocyclic analogs of the RXR selective modulator 1 (LG101506) were synthesized, and tested for their ability to bind RXRalpha and activate RXR homo and heterodimers. Potency and efficacy were observed to be dependent upon the choice of heterocycle as well as the sidechain employed.

Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that control diverse aspects of growth, development and homeostasis, making them exciting and important targets for drug discovery. In this review, some of the recent advances in our understanding of NRs, and their application to the discovery of new ligands, will be discussed.

A new series of hPPARalpha agonists containing a 2,4-dihydro-3H-1,2,4-triazol-3-one (triazolone) core is described leading to the discovery of 5 (LY518674), a highly potent and selective PPARalpha agonist.

Fluorinated trienoic acid analogues of the RXR selective modulator 1 (LG101506) were synthesized, and tested for their ability to bind RXRalpha and activate RXR homo and heterodimers. Potency and efficacy were observed to be dependent upon the position of fluorination, and improvement in pharmacological profile was demonstrated in some cases.

Retinoid X receptor:peroxisome proliferative-activated receptor (RXR:PPAR) heterodimers play a critical role in the regulation of glucose (RXR/PPARy) and lipid metabolism (RXR/PPARalpha). Previously, we described a concise structure-activity relationship study of selective RXR modulators possessing a (2E,4E,6Z)-3-methyl-7-(3,5-dialkyl-6-Eilkoxyphenyl)-octa-2,4,6-trienoic acid scaffold. These studies were focused on the 2-position alkoxy side chain. We describe here the design and synthesis of a novel series of RXR selective modulators possessing the same aromatic core structure with the addition of a ring locked 6--7-Z-olefin on the trienoic acid moiety. The synthesis and structure- activity relationship studies of these 6,7-locked cyclopentenyl, phenyl, thienyl, furan, and pyridine-trienoic acid derivatives is presented herein.